One hundred years ago, the first anecdotal observations correlating viral infection with tumor regression were reported, which lead to several investigations, over the course of the last century, evaluating the potential of various natural human and animal viruses to treat cancer. (Sinkovics et al., “New Developments in the Virus Therapy of Cancer: A Historical Review,” Intervirology 36:193-214 (1993)). These observations suggest that malignant lesions could regress in response to viral infection. To be effective in treatments of malignancies, such isolates should only grow productively and exhibit virulence in neoplastic cells, and should not be capable of propagating a productive infection through surrounding normal, terminally differentiated tissue. As virulence is governed by specific viral genes, one approach would be to genetically alter the virulence of viruses to obtain viruses which selectively destroy neoplastic cells. However, it was the achievements of the last two decades, most notably technical innovation in the area of molecular biology coupled with a heightened understanding of viral replication and pathogenesis at the genetic level, which ushered in the possibility of creating viruses in the laboratory which were selectively pathogenic for neoplastic cells. The advent of genetic engineering techniques has made it possible to selectively ablate viral virulence genes in the hope of creating a safe, avirulent virus that can selectively replicate in and destroy tumor cells. Such a virus would be able to propagate an infection throughout a tumor mass and directly kill the cancer cells, but be unable to inflict substantial damage to normal terminal differentiated cells. Although safe viruses have been created by such methodology, the attenuation process often has an overall deleterious effect on viral replication. This replication defect prevents the virus from completely destroying the tumor mass, and the surviving cancer cells can simply repopulate.
HSV-1 is a double-stranded DNA virus which is replicated and transcribed in the nucleus of the cell. HSV-1 consists of at least three groups of genes, α, β, and γ, whose expression is coordinately regulated and sequentially ordered in a cascade fashion. (Honess et al., “Regulation of Herpesvirus Macromolecular Synthesis, I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins,” J Vir 14:8-19 (1974)). Five immediate early (IE or α) genes are the first genes expressed during productive infection. While one of these genes encodes the immunomodulatory protein ICP47, the remaining four α genes encode infected cell proteins (ICPs) 0, 4, 22, and 27, the major regulatory proteins of the virus. These immediate early proteins then activate the expression of viral genes of the early (E or β) and late (L or γ) classes. Once the viral lifecycle advances beyond the IE stage, the ICP4 protein also autoregulates IE gene expression by reducing transcription from IE promoters (Preston, “Control of Herpes Simplex Virus Type 1 mRNA Synthesis in Cells Infected with Wild-Type Virus or the Temperature-Sensitive Mutant tsK,” J Vir 29: 275-284 (1979); Roberts et al., “Direct Correlation Between a Negative Autoregulatory Response Element at the Cap Site of the Herpes Simplex Virus Type1 IE175(α4) Promoter and a Specific Binding Site for the IE175 (α4) Protein,” J Vir 62: 4307-4320 (1988); Lium et al., “Repression of the alpha0 gene by ICP4 During a Productive Herpes Simplex Virus Infection,” J Vir 70: 3488-3496 (1996)). Proteins of the E class are responsible for viral DNA replication. The late (L) genes are induced after DNA replication and encode the structural components and enzymes required for assembly of virus particles.
Following infection of oral epithelial cells in its human host, HSV-1 invades axons and travels to the nuclei of sensory neurons that innervate this epithelia. Here, the virus establishes a latent infection, characterized by a restricted pattern of viral gene expression. (Roizman et al., “Herpes Simplex Viruses and Their Replication,” in: Knipe et al., eds. Fields Virology Vol. 2, 4th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, pp. 2399-2460 (2001)). Latency results in the permanent colonization of the host by the virus, and the severely limited expression of viral genes functions to shield the virus from host defenses.
In response to a variety of stimuli, these latent infections “reactivate,” resulting in episodes of productive viral growth characterized by expression of over 80 viral open reading frames (ORFs) distributed among two unique, single copy segments or within multiple repetitive loci of the large HSV-1 DNA genome. Activation of the productive or lytic gene expression program results in the production of viral particles and the eventual death of the infected cell. Distinct mRNA populations accumulate at discrete times in the productive replication cycle, resulting in the differential expression of viral genes in what has been termed a cascade pattern. (Roizman et al., “Herpes Simplex Viruses and Their Replication,” in: Knipe et al., eds. Fields Virology Vol. 2, 4th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, pp. 2399-2460 (2001)). The process is initiated by VP16, a transcription factor carried within the viral particle that recruits cellular transcription factors along with the RNA polymerase II holoenzyme to the promoters of the five viral IE genes. While one of these IE gene products dampens the host immune response by inhibiting the presentation of peptide antigens in conjunction with MHC class I molecules (Us12), the remaining four IE proteins are important for the subsequent expression of the next class of viral genes, the early or β genes. Viral early polypeptides primarily encode functions required for nucleotide metabolism and viral DNA synthesis, the initiation of which signals entry into the final late or γ phase of the viral life cycle. Two classes of late genes have been identified based upon their transcription in the presence of viral DNA synthesis inhibitors. While transcription of a subset of γ genes, the γ2 class, requires viral DNA synthesis, expression of γ1 genes is not completely dependent upon viral DNA replication and is only modestly reduced in the presence of inhibitors. Included among the late gene products are polypeptides critical for assembling infectious virus, virion components that function following entry but before IE gene expression, and proteins that regulate the host response to infection. Reactivation of a latent infection in a sensory neuron results in antereograde transport of viral progeny back to the portal of entry followed by the ensuing infection of epithelial cells, mobilization of the cellular immune response, and the formation of a fever blister or cold sore. Rarely, HSV-1 can enter and replicate within the CNS, causing encephalitis.
Martuza and colleagues were the first to demonstrate the therapeutic promise of an engineered oncolytic HSV-1 strain, the thymidine kinase (tk) negative HSV-1 mutant, dlsptk. (Martuza et al., “Experimental Therapy of Human Glioma by Means of a Genetically Engineered Virus Mutant,” Science 252:854-856 (1991)). tk mutants replicate effectively in actively dividing cells such as those found in tumors, but are relatively impaired for replication in non-dividing cells, such as neurons, and, therefore, display reduced neurovirulence compared with wild-type strains upon introduction into the CNS of adult mice. (Field et al., “The Pathogenicity of Thymidine Kinase-Deficient Mutants of Herpes Simplex Virus in Mice,” J Hyg (Lond) 81:267-277 (1978); Jamieson et al., “Induction of Both Thymidine and Deoxycytidine Kinase Activity by Herpes Viruses,” J Gen Virol 24:465-480 (1974); Field et al., “Pathogenicity in Mice of Strains of Herpes Simplex Virus Which are Resistant to Acyclovir In Vitro and In Vivo,” Antimicrob Agents Chemother 17:209-216 (1980); Tenser et al, “Trigeminal Ganglion Infection by Thymidine Kinase-Negative Mutants of Herpes Simplex Virus,” Science 205:915-917 (1979); Coen et al., “Thymidine Kinase-Negative Herpes Simplex Virus Mutants Establish Latency in Mouse Trigeminal Ganglia But Do Not Reactivate,” Proc Natl Acad Sci USA 86:4736-4740 (1989)). The tumor selected for oncolytic therapy was malignant glioma. The outcome for patients with this devastating brain tumor is grim, remaining essentially unchanged over the past 50 years despite advances in surgery, radiation, and chemotherapy. Direct injection of dlsptk into established tumors inhibited the growth of human glioma implants in athymic mice and prolonged survival of mice with intracranial gliomas as well. However, fatal encephalitis was still observed in 70-100% of the treated mice despite the fact that the tk mutant was significantly less neurovirulent than wild-type HSV-1. (Martuza et al., “Experimental Therapy of Human Glioma by Means of a Genetically Engineered Virus Mutant,” Science 252:854-856 (1991)). Thus, while it proved possible to use HSV-1 as an oncolytic virus to destroy cancer cells, the understanding of virulence was not sufficiently advanced to render the virus safe.
The breakthrough in creating attenuated HSV-1 strains resulted from characterizing viruses containing engineered mutations in the γ134.5 genes. Embedded within a repetitive genome component, the γ134.5 gene is expressed with γ1 late kinetics and is not required for growth in cultured monkey kidney cells (Vero cells). Strikingly, its impact on viral neurovirulence is greater than any single HSV-1 gene identified to date. (Chou et al., “Mapping of Herpes Simplex Virus-1 Neurovirulence to Gamma (1) 34.5, a Gene Nonessential for Growth in Culture,” Science 250:1262-1266 (1990); Maclean et al., “Herpes Simplex Virus Type 1 Deletion Variants 1714 and 1716 Pinpoint Neurovirulence-Related Sequences in Glasgow Strain 17+ Between Immediate Early Gene 1 and the ‘a’ Sequence,” J Gen Virol 72:631-639 (1991); Bolovan et al., “ICP34.5 Mutants of Herpes Simplex Virus Type 1 Strain 17syn+ Are Attenuated for Neurovirulence in Mice and for Replication in Confluent Primary Mouse Embryo Cell Cultures,” J Virol 68:48-55 (1994)). While the LD50 of many wild-type (WT) HSV-1 strains is less than 300 pfu following intracranial delivery, it is not possible to accurately measure the LD50 for γ34.5 mutant viruses. Indeed, upwards of 106-107 pfu of γ34.5 mutant viruses have been safely injected intracranially into mouse, non-human primate, and human brains (Chou et al., “Mapping of Herpes Simplex Virus-1 Neurovirulence to Gamma (1) 34.5, a Gene Nonessential for Growth in Culture,” Science 250:1262-1266 (1990); Maclean et al., “Herpes Simplex Virus Type 1 Deletion Variants 1714 and 1716 Pinpoint Neurovirulence-Related Sequences in Glasgow Strain 17+ Between Immediate Early Gene 1 and the ‘a’ Sequence,” J Gen Virol 72:631-639 (1991); Bolovan et al., “ICP34.5 Mutants of Herpes Simplex Virus Type 1 Strain 17syn+ Are Attenuated for Neurovirulence in Mice and for Replication in Confluent Primary Mouse Embryo Cell Cultures,” J Virol 68:48-55 (1994); Mineta et al., “Attenuated Multi-Mutated Herpes Simplex Virus-1 for the Treatment of Malignant Gliomas,” Nat Med 1:938-943 (1995); Hunter et al., “Attenuated, Replication-Competent Herpes Simplex Virus Type 1 Mutant G207: Safety Evaluation of Intracerebral Injection in Nonhuman Primates,” J Virol 73:6319-6326 (1999); Markert et al., “Conditionally Replicating Herpes Simplex Virus Mutant, G207 for the Treatment of Malignant Glioma: Results of a Phase 1 Trial,” Gene Ther 7:867-874 (2000); Rampling et al., “Toxicity Evaluation of Replication-Competent Herpes Simplex Virus (ICP 34.5 Null Mutant 1716) in Patients With Recurrent Malignant Glioma,” Gene Ther 7:859-866 (2000); Sundaresan et al., “Attenuated, Replication-Competent Herpes Simplex Virus Type 1 Mutant G207: Safety Evaluation in Mice,” J Virol 74:3832-3841 (2000)). In studies designed to examine the efficacy with which γ34.5 mutants were able to destroy human or murine gliomas implanted into mice, not only had the attenuation problem been solved, but the treated mice survived longer than their untreated counterparts and no longer developed viral encephalitis. Long-term surviving animals (usually in the vicinity of 60-80 days) were produced with efficiencies ranging from 10-50% of the treated animals depending on the tumor model and treatment regimen. (Markert et al., “Reduction and Elimination of Encephalitis in an Experimental Glioma Therapy Model with Attenuated Herpes Simplex Mutants That Retain Susceptibility To Acyclovir,” Neurosurgery 32:597-603 (1993); Chambers et al., “Comparison of Genetically Engineered Herpes Simplex Viruses for the Treatment of Brain Tumors in a SCID Mouse Model of Human Malignant Glioma,” Proc Natl Acad Sci USA 92:1411-1415 (1995); Kesari et al., “Therapy of Experimental Human Brain Tumors Using a Neuroattenuated Herpes Simplex Virus Mutant,” Lab Invest 73:636-648 (1995); Andreansky et al., “The Application of Genetically Engineered Herpes Simplex Viruses to the Treatment of Experimental Brain Tumors,” Proc Natl Acad Sci USA 93:11313-11318 (1996); Andreansky et al., “Evaluation of Genetically Engineered Herpes Simplex Viruses as Oncolytic Agents for Human Malignant Brain Tumors,” Cancer Res 57:1502-1509 (1997); Randazzo et al., “Treatment of Experimental Intracranial Murine Melanoma With a Neuroattenuated Herpes Simplex Virus 1 Mutant,” Virology 211:94-101 (1995) of Human Malignant Glioma,” Proc Natl Acad Sci USA 92(5):1411-1415(1995)). γ34.5 mutants were also tk+ and, therefore, retained their sensitivity to acyclovir, which could be used, if necessary, to control viral encephalitis. To further restrict viral replication to actively dividing cells, additional mutations in the UL39 ribonucleotide reductase gene or the UL2 uracil DNA glycosidase gene were introduced into the γ134.5 mutant background (Mineta et al., “Attenuated Multi-Mutated Herpes Simplex Virus-1 for the Treatment of Malignant Gliomas,” Nat Med 1:938-943 (1995); Kramm et al., “Therapeutic Efficiency and Safety of a Second-Generation Replication-Conditional HSV 1 Vector for Brain Tumor Gene Therapy,” Hum Gene Ther 8:2057-2068 (1997); Pyles et al., “A Novel Multiply-Mutated HSV-1 Strain for the Treatment of Human Brain Tumors,” Hum Gene Ther 8:533-544 (1997)). Although each of these non-neurovirulent, multi-mutated viruses could still reduce subcutaneous tumor growth and extend the survival of mice with intracranial tumors, more than 80% of the treated subjects still succumbed, emphasizing a different problem limiting the efficacy and outcome of treatment. While encephalitis was no longer observed in animals treated with any of these γ134.5 mutant derivatives, the γ134.5 deletion, either alone or in conjunction with additional mutations, impaired the replicative ability of these viruses in many human tumor cells, allowing the growth of residual glioma cells that ultimately killed the animals. Thus, the successful attenuation of HSV-1 left in its wake another problem for investigators to grapple with: engineered mutants that were sufficiently safe had lost a substantial amount of their replicative efficacy, impairing their oncolytic ability.
The reduced oncolytic ability of γ134.5 mutant derivatives results from their inability to counter an innate host defense designed to inhibit protein synthesis in virus-infected cells. After the initial reports established that the γ134.5 gene was a major determinant of HSV-1 neurovirulence non-essential for growth in cultured monkey kidney cells, further investigation revealed that γ134.5 mutants actually behaved like classical viral host range mutants, exhibiting restricted growth in some lines of cultured cells but not others. Thus, while a standard line of monkey kidney cells were permissive or supported the replication γ134.5 mutants, many human tumor cells were non-permissive, or did not support the growth of γ134.5 mutant derivatives. Upon infection of a non-permissive human tumor cell with a γ134.5 mutant strain, all of the events in the viral lifecycle proceeded normally up to and including viral DNA replication and the accumulation of γ2 late mRNA transcripts. These viral late mRNAs encoding key structural proteins required to complete the viral lifecycle and assemble the next generation of viral progeny, however, were never translated due to a block at the level of protein synthesis, effectively interrupting the viral lifecycle prior to the assembly and release of viral particles (Chou et al., “The Gamma (1) 34.5 Gene of Herpes Simplex Virus 1 Precludes Neuroblastoma Cells From Triggering Total Shutoff of Protein Synthesis Characteristic of Programmed Cell Death in Neuronal Cells,” Proc Natl Acad Sci USA 89:3266-3270 (1992)). Subsequent biochemical analysis demonstrated that the γ134.5 gene product was required to prevent accrual of phosphorylated eIF2, a critical translation initiation factor required to bring the initiator tRNA to the ribosome. Phosphorylation of eIF2 on its alpha subunit inactivates this translation factor and inhibits protein synthesis (Chou et al., “Association of a M(r) 90,000 Phosphoprotein With Protein Kinase PKR in Cells Exhibiting Enhanced Phosphorylation of Translation Initiation Factor eIF-2 Alpha and Premature Shutoff of Protein Synthesis After Infection With Gamma (1) 34.5-Mutants of Herpes Simplex Virus 1,” Proc Natl Acad Sci USA 92:10516-10520 (1995)). Thus, the inability of γ134.5 mutants to sustain protein synthesis in tumor cells limits their potential efficacy as replicating oncolytic viruses.
As obligate intracellular parasites, viruses are completely dependent upon the translational machinery resident in their host cells. It is not surprising, therefore, that a major innate host defense component centers on impeding viral mRNA translation. Indeed, the double-stranded RNA-dependent protein kinase PKR, an eIF2α kinase, is induced by the antiviral cytokines interferon α/β. It has been proposed that abundant dsRNA, a replicative intermediate formed in the replication of RNA viruses and a by-product of overlapping transcription units on opposite DNA strands of DNA viruses, is a signature of viral infection. PKR binds dsRNA and, in the presence of this activating ligand, forms a dimer, whereupon each subunit phosphorylates the other. It is this activated, phosphorylated form of PKR that then goes on to phosphorylate other substrates, including eIF2α, the regulatory subunit of eIF2 (Kaufman R J, “Double-Stranded RNA-Activated Protein Kinase PKR,” In: Sonenberg eds. Translational Control. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 503-528 (2000)). Should cells initially infected succeed in inhibiting translation, the viral invader would effectively be stopped in its tracks, denied access to the cellular translational apparatus it needs to complete its life-cycle. This arm of the innate host response, then, is designed to sacrifice the initially infected cells for the benefit of the larger population. Any effective oncolytic virus must be able to thwart host defenses if it is to propagate an infection throughout a tumor causing regression and, ultimately, the destruction of the tumor. Failure to mount an effective response against both innate and acquired host defenses is likely to prevent viral replication and subsequent spread throughout the tumor tissue. The present invention describes how to engineer an attenuated γ134.5 mutant virus that is capable of countering both the acquired immune response and the innate host responses mediated by interferon.
At least two herpes simplex virus gene products have been implicated in the virus's ability to resist the pleiotropic effects of interferon (IFN). One of these is the product of the ICP0 gene, a multifunctional polypeptide produced very early in the viral life cycle that transactivates viral gene expression (Everett, R. D., “ICP0, A Regulator of Herpes Simplex Virus During Lytic and Latent Infection,” Bioessays 22:761-770 (2000)). ICP0 deficient mutants are hypersensitive to IFN, as viral mRNAs do not accumulate in IFN treated Vero cells, whereas cellular mRNAs encoding IFN induced gene products increase in abundance (Eidson et al., “Expression of Herpes Simplex Virus ICP0 Inhibits the Induction of Interferon-Stimulated Genes by Viral Infection,” J Virol 76:2180-2191 (2002), Mossman et al., “Herpes Simplex Virus Triggers and Then Disarms a Host Antiviral Response,” J Virol 75:750-758 (2001), Mossman et al., “Herpes Simplex Virus ICP0 and ICP34.5 Counteract Distinct Interferon-Induced Barriers To Virus Replication,” J Virol 76:1995-1998 (2002), Nicholl et al., “Activation of Cellular Interferon-Responsive Genes After Infection of Human Cells With Herpes Simplex Type 1,” J Gen Virol 81:2215-2218 (2000)). This particular IFN antiviral effect requires the cellular promyelocytic leukemia (PML) gene product, and it has been proposed that the disassembly of PML bodies observed in HSV-1 infected cells, which requires the ubiquitin E3 ligase activity of ICP0 (Boutell et al., “Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 And Its Isolated RING Finger Domain Act As Ubiquitin E3 Ligases in vitro,” J Virol 76:841-850 (2002), Van Sant et al., “The Infected Cell Protein 0 of Herpes Simplex Virus 1 Dynamically Interacts With Proteasomes, Binds And Activates The cdc34 E2 Ubiquitin-Conjugating Enzyme, And Possesses In Vitro E3 Ubiquitin Ligase Activity,” Proc Natl Acad Sci USA 98:8815-20 (2001)), enables the virus to prevent the induction of IFN responsive genes (Chee et al., “Promyelocytic Leukemia Protein Mediates Interferon-Based Anti-Herpes Simplex Virus 1 Effects,” J Virol 77:7101-7105 (2003)). While the ICP0 polypeptide prevents the transcriptional induction of cellular IFN responsive genes, the γ134.5 gene, when altered, results in an IFN hypersensitive virus, encodes a product that operates by preventing host defenses from inactivating the critical translation initiation factor eIF2 (Cerveny et al., “Amino Acid Substitutions In The Effector Domain of The γ134.5 Protein of Herpes Simplex Virus I Have Differential Effects On Viral Response To Interferon-α,” Virology 307:290-300 (2003), Cheng et al., “Val193 and Phe195 of The γ134.5 Protein of Herpes Simplex Virus 1 Are Required For Viral Resistance To Interferon α/β,” Virology 290:115-120 (2001), Mossman et al., “Herpes Simplex Virus ICP0 and ICP34.5 Counteract Distinct Interferon-Induced Barriers To Virus Replication,” J Virol 76:1995-1998 (2002)).
Upon binding the catalytic subunit of protein phosphatase1α (PP1α), the γ134.5-PP1α holoenzyme prevents the accumulation of phosphorylated, inactive eIF2α in infected cells, preserving viral translation rates (He et al., “The Gamma(1)34.5 Protein of Herpes Simplex Virus 1 Complexes With Protein Phosphatase 1 Alpha To Dephosphorylate The Alpha Subunit of Eukaryotic Initiation Factor 2 And Preclude The Shutoff of Protein Synthesis By Double-Stranded RNA-Activated Protein Kinase,” Proc Natl Acad Sci USA 94:843-848 (1997)). However, in many established human cell lines infected with a γ134.5 mutant virus, the onset of viral DNA synthesis and the accumulation of γ2 late viral mRNA transcripts are accompanied by the complete cessation of cellular and viral protein synthesis (Chou et al., “The γ34.5 Gene of Herpes Simplex Virus 1 Precludes Neuroblastoma Cells From Triggering Total Shutoff of Protein Synthesis Characteristic of Programmed Cell Death In Neuronal Cells,” Proc Natl Acad Sci USA 89:3266-3270 (1992)). Thus, γ134.5 mutants are not only deficient in functions intrinsic to the γ134.5 gene product, but, by failing to translate the viral γ2 mRNAs, they are also deficient in all the activities encoded by this entire class of genes as well. Importantly, when dealing with phenotypes ascribed to a deficiency in the γ134.5 gene, it is fair to question whether the failure to translate these late γ2 viral mRNAs contributes to the observed phenotype. One of these late γ2 mRNAs encodes the Us11 polypeptide, a dsRNA binding (Khoo et al., “Characterization of RNA Determinants Recognized by The Arginine- and Proline-Rich Region of Us11, A Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation,” J Virol 76:11971-11981 (2002)), ribosome-associated protein (Roller et al., “The Herpes Simplex Virus 1 RNA Binding Protein Us11 Is A Virion Component And Associates With 60S Ribosomal Subunits,” J Virol 66:3624-3632 (1992)) that physically associates with PKR (Cassady et al., “The Herpes Simplex Virus Type 1 Us11 Protein Interacts With Protein Kinase R In Infected Cells and Requires a 30 Amino Acid Sequence Adjacent to a Kinase Substrate Domain,” J Virol 76:2029-2035 (2002); Poppers et al., “Identification of a Lytic-Cycle Epstein-Barr Virus Gene Product That Can Regulate PKR Activation,” J Virol 77:228-236 (2003)) and can prevent PKR activation in response to dsRNA and PACT, a cellular protein that can activate PKR in an RNA independent manner (Peters et al., “Inhibition of PACT-Mediated Activation of PKR By The Herpes Simplex Virus Type 1 Us11 Protein,” J Virol 76:11054-11064 (2002)). Furthermore, Us11 can preclude the premature cessation of protein synthesis observed in cells infected with a γ134.5 mutant when it is expressed at immediate-early, as opposed to late times, post-infection (Mohr et al., “A Herpesvirus Genetic Element Which Affects Translation In The Absence of The Viral GADD34 Function,” EMBO J 15:4759-4766 (1996); Mulvey et al., “A Herpesvirus Ribosome Associated, RNA-Binding Protein Confers A Growth Advantage Upon Mutants Deficient in a GADD34-Related Function,” J Virol 73:3375-3385 (1999)). Recently, it was demonstrated that the premature cessation of translation observed in cells infected with a γ134.5 mutant actually results from the combined loss of γ134.5 function along with the failure to translate the Us11 mRNA, establishing that HSV-1 utilizes different mechanisms to regulate eIF2α phosphorylation at discrete phases of the viral life cycle (Mulvey et al., “Regulation of eIF2α Phosphorylation By Different Functions That Act During Discrete Phases In The HSV-1 Lifecycle,” J Virol 77:10917-10928 (2003)).
Numerous replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral γ34.5 virulence gene have been used as oncolytic agents. (U.S. Pat. Nos. 5,328,688 and 6,071,692 to Roizman; U.S. Pat. No. 5,824,318 to Mohr et al.; Mulvey et al., “Regulation of eIF2α Phosphorylation By Different Functions That Act During Discrete Phases In The HSV-1 Lifecycle,” J Virol 77:10917-10928 (2003); (Taneja et al., “Enhanced Antitumor Efficacy of a Herpes Simplex Virus Mutant Isolated by Genetic Selection in Cancer Cells,” Proc Natl Acad Sci USA 98:8804-08 (2001); Markert et al., “Genetically Engineered HSV in the Treatment of Glioma: A Review,” Rev Med Virol 10(1):17-30 (2000); Martuza et al., “Conditionally Replicating Herpes Vectors for Cancer Therapy,” J Clin Invest 105(7):841-846 (2000); Andreansky et al., “The Application of Genetically Engineered Herpes Simplex Viruses to the Treatment of Experimental Brain Tumors,” Proc Natl Acad Sci USA 93(21): 11313-8 (1996); Kesari et al., “Therapy of Experimental Human Brain Tumors Using a Neuroattenuated Herpes Simplex Virus Mutant,” Lab Invest 73(5):636-648 (1995); Mineta et al., “Attenuated Multi-Mutated Herpes Simplex Virus-1 for the Treatment of Malignant Gliomas,” Nature Medicine 1(9):938-43 (1995); Chambers et al., “Comparison of Genetically Engineered Herpes Simplex Viruses for the Treatment of Brain Tumors in SCID Mouse Model of Human Malignant Glioma,” Proc Natl Acad Sci USA 92(5):1411-1415(1995)). However, a major limitation in the use of attenuated, replication-competent viruses to directly destroy tumors continues to be the reduced growth of these weakened strains in many cell types, including cancer cells. Despite an initial wave of oncolysis, host defenses trigger an inability of the viral vector to replicate successfully for long enough to eradicate the entire population of neoplastic cells, and the surviving cancer cells re-establish their strangle-hold on the patient.
What is needed now is a thorough understanding of the contribution made by the Us11 gene towards the overall IFN-resistant phenotype of HSV-1, and the application of that information to the making of improved, more efficacious viral anti-tumor agents.
The present invention is directed to overcoming these and other deficiencies in the art.